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    • #14937
      kutya7890
      Participant

      Hi guys,

      i have a question, might be very stupid. I wanna generate a mouse which is a reporter so after my gene of interest there would be ires gfp. my question is that the CDS contains the stop codon at the end but the mRNA continues. where do i insert my ires gfp. dierctly after the stop codon of the CDS or i add additional stop codons after the end of the mRNA?
      thanks for input.

    • #104856
      JackBean
      Participant

      so, you want to create fused protein? If I understood correctly, you want only transcriptional fusion, rigth? I’ve just recetly read an article about that, so you might find it useful, although it’s about plants
      http://www.ncbi.nlm.nih.gov/pubmed/19568949

    • #104865
      kutya7890
      Participant

      thanks.
      yes, it would be a fusion protein. I have my gene of interest, at the end of the CDS there is a stop codon, later i wanna insert an IRES-egfp-cre-ert2 triple fusion protein. I was wondering whether in the 3’UTR insertion my fusion protein disturbes the mRNA of my native protein or it is fine. What is the general lenght between the stop codon and the ires?

    • #104877
      JackBean
      Participant

      Well, you write it the other way. So let’s make it clear.
      There are 2 possibilities, either translational fusion or transcriptional fusion.
      In the first one you have to have only 1 mRNA, 1 ORF, which results in only 1 polypeptide, which consists of 2 proteins.
      In the second one, you have 1 mRNA, but you have 2 distinctive ORFs separated by their START and STOP codons and thus you get two distinctive proteins.

      Which one you want? You wrote about triple fusion protein, so that means the second one?

    • #104890
      kutya7890
      Participant

      sorry if it was not clear.
      the construct will look like this (at least that is what i hope)
      5′ gene of interest-stop-ires-egfp-cre-ert2-3′ utr’
      so this is one mrna, two proteins, and the second one is a triple fusion protein.
      the question is: the stop codon is at the and of the cds where should i place my ires? directly after or with some space?
      hope it is clear now!

    • #104894
      JackBean
      Participant

      OK, so you’re doing both translational and transcriptional fusion 😆 😆 😆
      There should be probably some little space (~20nt), check the article I’ve send you above.

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